Antibodies to peptides having NGF-like activity, and use thereof

ABSTRACT

Disclosed are (A) an antibody to a peptide consisting of 8 to 9, or 12 to 14 successive amino acids from a polypeptide having nerve growth factor-like activity, (B) a method for producing the antibody, (C) a cloned hybridoma which produces the antibody, (D) a method for producing the cloned hybridoma, (E) a conjugate of the above mentioned peptide with a carrier protein, and (F) a method for purifying, and a method for detecting and assaying a polypeptide having nerve growth factor-like activity.

This is a continuation of application Ser. No. 08/350,214, filed Dec. 6,1994, which is a CIP of Ser. No. 08/041,904, filed on Apr. 2, 1993, nowabandoned, which is a continuation of Ser. No. 07/574,431, filed Aug.29, 1990, (abandoned).

BACKGROUND OF THE INVENTION

The present invention relates to an antibody to a peptide including atleast 8 successive amino acids of an amino acid sequence represented byformula 1! described below, and relates to hybridomas, methods forproducing the same and uses thereof.

Many cell growth factors have been isolated and their structures havebeen elucidated since the discovery of epidermal growth factor(hereinafter referred to as EGF) and nerve growth factor (hereinafterreferred to as NGF).

Cell growth factors are useful for the elucidation of celldifferentiation mechanism and cell proliferation mechanism, and some ofthem, including human EGF, are expected to be useful as medicines.Therefore, studies thereon have become increasingly prevalent in recentyears.

As to human NGF, its genomic DNA has been isolated, but has not beenexpressed in a host cell. Hence, the investigations of producing humanNGF in large amounts and of using it have not been advanced.

The present inventors cloned a cDNA sequence coding for a polypeptide(I) which showed about 60% homology to human NGF from human glioma cDNAlibraries (Japanese Patent Unexamined Publication No. 1-193654/1989which corresponds to EP Application No. 90 104 419.8 and U.S. patentapplication Ser. No. 07/488,696, refer to FIGS. 1 to 4). The polypeptide(I) includes the following amino acid sequence represented by formula 1!in a molecule thereof: ##STR1## The polypeptide (I) is considered tohave an action similar to that of NGF and important roles in vivo suchas enhancement of differentiation, growth and proliferation of animalcells, enhanced expression of genes and induction of proteins andenzymes. This polypeptide has therefore the high possibility that it canbe used as medicines.

Basic information as to the polypeptide (I) such as the distributionthereof in vivo, the production mode thereof or the mechanism ofactivity expression favors the development of the polypeptide (I) asmedicines.

It is also important to know the amount of the polypeptide (I) exactly,when this protein is purified from a gene recombinant.

Previously, the amount of NGF has been calculated by assaying theneurite outgrowth action to PC12 cells. Also, the neurite outgrowthaction to avian dorsal root ganglia has been utilized to calculate theamount of NGF. However, these assays have the disadvantages that theelaborate procedure is required and the measurement error is large, inaddition, that a long time is required to obtain the result. For thesereasons, it has been desired to develop means of assaying thepolypeptide (I) simply and exactly.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide means ofassaying the polypeptide (I) simply and exactly. Other objects will beapparent from the following description and appended drawings.

The present invention provides:

(1) an antibody to a peptide including at least 8 successive amino acidsof an amino acid sequence represented by formula 1! wherein saidantibody has no substantial cross-reactivity with human NGF,

(2) the antibody described in (1), in which the antibody is a polyclonalantibody,

(3) the antibody described in (1), in which the antibody is a monoclonalantibody,

(4) the antibody described in (1), (2) or (3), in which the antibody isobtained by using a conjugate of a peptide with a carrier protein as animmunogen, the peptide including at least 8 successive amino acids ofthe amino acid sequence represented by formula 1!,

(5) the antibody described in (1) or (4), in which the peptide is apolypeptide (I) including the amino acid sequence represented by formula1!,

(6) the antibody described in (1) or (4), in which the peptide

is a partial peptide of the polypeptide (I), the partial peptideconsisting of 12 to 14 successive amino acids of a sequence representedby formula 2!, Tyr Ala Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys,or 8 to 9 successive amino acids of a sequence represented by formula3!, Cys Ala Leu Ser Arg Lys Ile Gly Arg,

(7) a method for producing the polyclonal antibody described in (2)which comprises immunizing a mammal with a peptide including at least 8successive amino acids of the amino acid sequence represented by formula1! or with a conjugate of the peptide with a carrier protein to form thepolyclonal antibody, and then collecting the polyclonal antibody,

(8) a method for producing the monoclonal antibody described in (3)which comprises proliferating a cloned hybridoma composed of a spleencell of a mammal and a lymphoid cell of the mammal in a liquid culturemedium or in an peritoneal cavity of the mammal to form and accumulatethe monoclonal antibody, the spleen cell of the mammal being immunizedwith a peptide including at least 8 successive amino acids of the aminoacid sequence represented by formula 1! or with a conjugate of thepeptide with a carrier protein, and then collecting the monoclonalantibody,

(9) A cloned hybridoma composed of a spleen cell of a mammal and alymphoid cell of the mammal, the spleen cell of the mammal beingimmunized with a peptide including at least 8 successive amino acids ofthe amino acid sequence represented by formula 1! or with a conjugate ofthe peptide with a carrier protein,

(10) a method for producing a cloned hybridoma composed of a spleen cellof a mammal and a lymphoid cell of the mammal, which comprises cellfusing the spleen cell of the mammal with the lymphoid cell thereof toform a fused cell, the spleen cell of the mammal being immunized with apeptide including at least 8 successive amino acids of the amino acidsequence represented by formula 1! or with a conjugate of the peptidewith a carrier protein, and then cloning the fused cell,

(11) a partial peptide of the polypeptide (I), the partial peptideconsisting of 12 to 14 successive amino acids of the sequencerepresented by formula 2!, Tyr Ala Glu His Lys Ser His Arg Gly Glu TyrSer Val Cys.

(12) a partial peptide of the polypeptide (I), the partial peptideconsisting of 8 to 9 successive amino acids of the sequence representedby formula 3!, Cys Ala Leu Ser Arg Lys Ile Gly Arg,

(13) a conjugate of a peptide including at least 8 successive aminoacids of the amino acid sequence represented by formula 1! with acarrier protein,

(14) the conjugate described in (13), in which the peptide is thepolypeptide (I) including the amino acid sequence represented by formula1! in a molecule thereof,

(15) the conjugate described in (13), in which the peptide is thepartial peptide described in (11) or (12),

(16) a method for purifying the polypeptide (I) which comprises usingthe antibody described in (1), (3), (4), (5) or (6), and

(17) a method for detecting and assaying the polypeptide (I) whichcomprises using the antibody described in (1), (3), (4), (5) or (6).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction enzyme map of a DNA comprising a polypeptide (I)cDNA contained in plasmid pUNK5 obtained in Reference Example 1;

FIG. 2 shows a nucleotide sequence of the DNA containing the polypeptide(I) cDNA contained in plasmid pUNK5 obtained in Reference Example 1, andan amino acid sequence translated therefrom;

FIG. 3 shows a comparison of the amino acid sequence (the upper row) ofthe polypeptide (I) of the present invention described in ReferenceExample 1 with an amino acid sequence (the lower row) of human NGF;

FIG. 4 is a restriction enzyme map of a DNA comprising a polypeptide (I)cDNA contained in plasmid pHNT2 obtained in Reference Example 2;

FIG. 5 shows a nucleotide sequence of the DNA sequence comprising thepolypeptide (I) cDNA contained in plasmid pHNT2 obtained in ReferenceExample 2, and an amino acid sequence translated therefrom;

FIG. 6 is a schematic representation showing the construction ofpolypeptide (I) expression vector pENGFT102 for Escherichia coliobtained in Reference Example 3;

FIG. 7 is a schematic representation showing the construction ofpolypeptide (I) expression vectors pNTK26 and pNTL145 for animal cellsobtained in Reference Example 5;

FIG. 8 is a schematic representation showing the construction ofpolypeptide (I) expression vector pNTS101 for animal cells obtained inReference Example 6;

FIG. 9 is a graph showing the results of determination of thepolypeptide (I) by EIA in Example 7, using monoclonal antibody MoAb82-4obtained in Example 5 and HRP-MoAb4-2 obtained in Example 6;

FIG. 10 is a graph showing the results of competitive binding inhibitionof various peptides to monoclonal antibody MoAb4-2, obtained in Example8;

FIG. 11 is a graph showing the results of competitive binding inhibitionof various peptides to monoclonal antibody MoAb46-31, obtained inExample 8;

FIG. 12 is a graph showing the results of compeptitive bindinginhibition of various peptides to monoclonal antibody MoAb82-4, obtainedin Example 8;

FIG. 13 is a graph showing the results of compeptitive bindinginhibition of various peptides to monoclonal antibody MoAb84-62,obtained in Example 8; and

FIG. 14 is a diagram showing the results of Western blotting analysisusing monoclonal antibody MoAb4-2 obtained in Example 5.

DESCRIPTION OF THE PREFERRED EMBODIMENT

When the antibodies of the present invention are produced, thepolypeptide (I) or its partial peptides can be used as immunogens.

The polypeptide (I) of the present invention includes a polypeptidehaving the amino acid sequence of formula 1! and a polypeptide furtherhaving a threonine residue at the C-terminus of the amino acid sequenceof formula 1!. Further, the polypeptide (I) of the present inventionincludes a polypeptide having several amino acid residues added to theN-terminus and/or the C-terminus of the amino acid sequence of formula1!.

In Reference Examples described below, the polypeptide (I) having thefollowing amino acid sequence represented by formula 1'! in which Thr isadded to the C-terminus of the amino acid sequence represented byformula 1! was produced by expression in Escherichia coli. ##STR2##

Further, in Reference Examples described below, the polypeptide (I)expressed in animal cells is believed to have the amino acid sequencerepresented by formula 1! or 1'!

In addition to the polypeptides described above, the polypeptide (I) ofthe present invention includes portions of the above polypeptides whichhave the same activity as the above polypeptides, and polypeptides inwhich portions of the above amino acid sequences are replaced with oneor more different amino acids or amino acid sequences, or in which oneor more different amino acids or amino acid sequences is added to orinserted into the above amino acid sequences, and which have the sameactivity as the above polypeptides.

When the polypeptide (I) is produced by using gene recombinanttechniques, a methionine residue corresponding to initiation codon ATGupstream from a gene coding for the polypeptide (I) may be added to theN-terminus of the polypeptide (I).

The polypeptide (I) is obtained, for example, by introducing anexpression vector containing a DNA coding for the polypeptide (I) intoan appropriate host and then cultivating the resulting transformant.

The above expression vector containing a nucleotide sequence coding forthe polypeptide (I) can be obtained, for example, by the followingprocess:

(a) RNA coding for polypeptide (I) is isolated.

(b) Single stranded complementary DNA (cDNA) is synthesized from theRNA, followed by synthesis of double stranded DNA.

(c) The complementary DNA is introduced into a plasmid.

(d) A host cell is transformed with the recombinant plasmid thusobtained.

(e) After culturing the transformants thus obtained, the plasmidcontaining the desired DNA is isolated from the transformants by anappropriate method such as colony hybridization using a DNA probe.

(f) The desired cloned DNA is cut out from the plasmid.

(g) The cloned DNA is ligated downstream from a promoter in the vector.

The RNA coding for the polypeptide (I) can be obtained from variouspolypeptide (I)-producing cells, for example, human glioma cells,pituicytes and fibroblasts.

The expression vector thus obtained is introduced into appropriate hostcells (such as Escherichia coli, Bacillus subtilis, yeast and animalcells) and the resulting transformants are cultivated, whereby thepolypeptide (I) can be produced.

Partial peptides containing at least 8 successive amino acids of theamino acid sequence of the polypeptide (I) can be used as immunogens.Examples of such partial peptides include N-terminal partial peptidesconsisting of 12 to 14 successive amino acids of a sequence representedby formula 2!, Tyr Ala Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cysand C-terminal partial peptides consisting of 8 to 9 successive aminoacids of a sequence represented by formula 3!, Cys Ala Leu Ser Arg LysIle Gly Arg.

The above partial peptides can be produced by peptide synthesis methodsknown in the art, which may be any of the known solid phase synthesismethods and liquid phase synthesis methods. Examples of such peptidesynthesis methods include the methods described in Schroder and Lubke,The Peptide, Vol. 1, Academic Press, New York, U.S.A. (1966), Izumiya etal., Peptide Synthesis, Maruzen (1975) and Izumiya et al., Fundamentalsand Experiments of Peptide Synthesis, Maruzen (1985).

The partial peptides may be produced by cleaving the polypeptide (I)with appropriate enzymes. Such methods include, for example, the methoddescribed in Course of Biochemical Experiments 1, Chemistry of Proteins,pages 255 to 332, edited by Biochemical Society of Japan, Tokyo KagakuDojin (1976).

When peptides including at least 8 successive amino acids of an aminoacid sequence represented by formula I! are used for immunization, theymay be used as conjugates with carrier proteins.

Examples of the carrier proteins include bovine serum albumin, bovinethyroglobulin, bovine γ-globulin, hemocyanin, and Freund's completeadjuvant (Difco).

The above peptides can be coupled with the carrier proteins by methodsknown in the art. Reagents used for the coupling include, for example,glutaraldehyde and water-soluble carbodiimide. The weight ratio of thepeptide to the carrier protein is suitably about 1:1 to 1:30, preferablyabout 1:15 to 1:20, more preferably about 1:1 to 1:4. When the reactionis conducted at a pH around neutrality, particularly of approximately7.3, good results are obtained in many cases. The time required for thereaction is preferably about 2 to 6 hours in many cases, more preferablyabout 3 hours. The conjugates thus prepared are dialyzed against waterat about 4° C. by conventional methods. The resulting products may bestored frozen or lyophilized.

In order to produce the polyclonal antibodies, warm-blooded animals areinoculated with the immunogens prepared as described above. Examples ofthe warm-blooded animals used for production of the above antibodiesinclude warm-blooded mammals (such as sheep, goat, rabbits, bovines,rats, mice, guinea pigs, horses and pigs) and birds (such as chickens,pigeons, ducks, geese and quail). The immunogens are inoculated into thewarm-blooded animals in amounts effective for antibody production. Forexample, 1 mg of the immunogen per one inoculation is emulsified with 1ml of physiological saline and Freund's complete adjuvant, andsubcutaneously inoculated into the back and hind-limb palm of the rabbitevery 4 weeks, 5 times in total, whereby the antibody is produced inmany cases. The antibodies thus formed in the warm-blooded animals arecollected. For example, in case of the rabbit, the blood is collectedfrom the aural vein usually 7 to 12 days after the final inoculation andsubjected to centrifugation to obtain the antibody as a serum. Theresulting antiserum is usually subjected to affinity chromatographyusing a carrier bearing each antigen peptide to recover adsorbedfractions, whereby the polyclonal antibody can be purified.

On the other hand, the monoclonal antibody can also be utilized which isobtained by a method similar to that of Milstein et al. Nature 256, 495(1975)!. Namely, the monoclonal antibody-producing hybridoma cells ofthe present invention can be prepared by selecting individuals showing ahigh antibody titer from the warm-blooded animals, for example, mice,immunized similarly with the above method for preparing the polyclonalantibody, collecting spleens or lymphatic corpuscles therefrom after 2to 5 days from the final immunization, and fusing antibody-producingcells contained therein with myeloma cells. The fusing procedure can beconducted according to methods known in the art, for example, the methodof Kohler and Milstein Nature 256, 495 (1975)!. Fusion accelerators,including polyethylene glycol (PEG) and Sendai virus, may be used. Inparticular, PEG is preferably used. Examples of the myeloma cellsinclude NS-1, P3U1 and SP2/0, and particularly P3U1 is preferably used.The ratio of the number of the antibody-producing cells to the number fthe myeloma cells is preferably about 1:1 to 20:1. PEG (preferably PEG1,000 to PEG 6,000) is added in a concentration of about 10 to 80%,followed by incubation at 20° to 40° C., preferably 30° to 37° C., for 1to 10 minutes, whereby cell fusion can be effectively performed.

In order to obtain the monoclonal antibodies of the present invention,it is preferred to use rats or mice for immunization. When the mice areimmunized, for example, subcutaneous, intraperitoneal or intravenousinjections are preferably used. In particular, subcutaneous injection ispreferable. The immunizing interval and the immunizing dose are widelychangeable, and various methods are available. For example, methods inwhich immunization is carried out about 2 to 6 times at intervals of 2weeks and spleen cells are removed after about 1 to 5 days, preferablyabout 2 to 4 days from the final immunization are frequently used. Asthe immunizing dose, it is preferred to use about 0.1 μg or more,preferably about 10 to 300 μg of the peptide per one immunization.Further, it is desirable to carry out the fusion process using thespleen cells after confirmation of an increase in antibody titer inblood by collecting a portion of blood before removal of the spleens.

The above spleen cells are fused with lymphoid cells. For example, thespleen cells removed from the mice are fused with lymphoid cell strainssuch as suitable myeloma cells for example, P3-X-63-Ag 8UI (Ichimori etal., J. Immun. Method 80, 55 (1985))! of the same kind or a differentkind (preferably the same kind) having markers such ashypoxanthine-guanine-phosphoribosyl-transferase deficient (HGPRT⁻) andthymidine kinase deficient (TK⁻). For example, the fused cells areproduced in accordance with the method of Kohler and Milstein Nature256, 495 (1975)!. For example, myeloma cells and spleen cells in a ratioof about 1:5 are suspended in a medium (hereinafter referred to as IHmedium) prepared by mixing Iscove medium and Ham F-12 medium in a 1:1ratio, and a fusion accelerator such as Sendai virus or polyethyleneglycol (PEG) is added thereto. It is of course possible to add otherfusion accelerators such as dimethyl sulfoxide (DMSO). Thepolymerization degree of PEG is usually about 1,000 to 6,000, the fusiontime is about 0.5 to 30 minutes, and the concentration of the suspensionis about 10 to 80%. As a preferred condition, the myeloma cells and thespleen cells are fused with each other in a concentration of about 35 to55% for about 4 to 10 minutes using PEG 6,000, which results inefficient fusion. The fused cells can be selectively proliferated usinghypoxanthine-aminopterin-thymidine medium (HAT medium) Nature 256, 495(1975)!.

The culture supernatant of the proliferated cells is then screened fordesired antibody production. The screening of the antibody titer can becarried out in the following manner. First, the presence or absence ofthe antibody produced by peptide immunization is examined by radioimmunoassays (RIAs) or enzyme immunoassays (EIAs). For these methods,various modified methods are also available. As a preferred example ofthe assays, a method using the EIA is hereinafter described. A rabbitanti-mouse immunoglobulin antibody is coupled with a carrier such ascellulose beads according to conventional methods, and then a culturesupernatant or mouse serum to be assayed is added thereto, followed byreaction at a constant temperature (about 4° to 40° C., the same appliedhereinafter) for a definite time. After the reaction product isthoroughly washed, an enzyme-labeled peptide (a peptide is coupled withan enzyme according to conventional methods, followed by purification)is added thereto, followed by reaction at a constant temperature for adefined time. After the reaction product is thoroughly washed, an enzymesubstrate is added thereto, followed by reaction at a constanttemperature for a defined time. Then, the absorbance or fluorescence ofthe colored product is measured.

It is desirable that the cells in wells which show cell proliferation ina selective medium and antibody activity to the peptide used forimmunization are cloned by a limiting dilution analysis. The supernatantof the cloned cells is similarly screened to increase the cells in thewells which show a high antibody titer, whereby monoclonalantibody-producing hybridoma clones showing the reactivity with theimmunized peptide can be obtained.

The hybridoma cells thus cloned are proliferated in a liquid medium.Specifically, for example, the hybridoma cells are cultivated in theliquid medium such as a medium prepared by adding about 0.1-40% bovineserum to RPMI-1640 G. E. Moore et al., J. Am. Med. Assoc. 199, 549(1967)!, for about 2 to 10 days, preferably 3 to 5 days, whereby themonoclonal antibody can be obtained from the culture solution. Theantibody can further be obtained by intraperitoneally inoculatingmammals with the hybridoma cells thereby proliferating the cells andthen collecting the ascites. In the case of mouse, for example, about1×10⁴ to 1×10⁷, preferably 5×10⁵ to 2×10⁶ of the hybridoma cells areintraperitoneally inoculated into a mouse such as BALB/c preliminarilyinoculated with mineral oil and the like, and the ascites is collectedafter about 7 to 20 days, preferably after about 10 to 14 days. Themonoclonal antibody formed and accumulated in the ascites can be easilyisolated as pure immunoglobulin by ammonium sulfate fractionation,DEAE-cellulose column chromatography or the like.

The monoclonal antibodies which specifically bind to the peptideincluding at least 8 seccessive amino acids of an amino acid sequencerepresented by formula 1! are thus obtained.

The monoclonal antibodies of the present invention specifically bind toimmunogens, the peptide including at least 8 seccessive amino acids ofan amino acid sequence represented by formula 1!.

In some cases, the monoclonal antibodies of the present invention bindto the peptide including at least 8 successive amino acids of an aminoacid sequence represented by formula 1! which is different from thepeptide used as an immunogen when the antibodies are produced.

The monoclonal antibodies of the present invention are monoclonalantibodies to peptides including at least 8 successive amino acids of anamino acid sequence represented by formula 1! which are immunogenpeptides.

The monoclonal antibodies of the present invention have the property ofspecifically binding to the peptide including at least 8 seccessiveamino acids of an amino acid sequence represented by formula 1!.

The monoclonal antibodies of the present invention have a molecularweight of about 140 to 16 kilodaltons and belong to IgM or IgG inimmunoglobulin class.

The molecules of the above antibodies may be fractions thereof, such asF(ab')₂, Fab' or Fab. As the molecule to which a labeling agentdescribed below is directly bound, Fab' is preferred.

The monoclonal antibodies of the present invention are very useful asreagents for assaying the polypeptide (I), because they specificallybind to the polypeptide (I) and have no substantial cross-reactivitywith human NGF. It is also very useful from the viewpoint of obtainingbasic information (such as distribution in vivo) regarding thepolypeptide (I) to make it easy to assay the polypeptide (I) in livingorgans and tissues. In order to detect the polypeptide (I) in the livingorgans and tissues, enzyme immunoassays (ELAs), fluorescent antibodymethods and radio immunoassays (RIAs) are usually employed. Westernblotting analysis may be used to determine the size of the polypeptide(I) in these organs and tissues. In this method, crude extracts derivedfrom the organs or the tissues or partially purified samples thereof aresubjected to polyacrylamide gel electrophoresis, followed by transfer tomembrane filters to detect the polypeptide (I) with HRP-boundantibodies.

For the antibodies having neutralizing activity, it is also possible topursue the function of the polypeptide (I) in vivo by neutralizing theactivity of the polypeptide (I).

It is further possible to use antibody affinity columns prepared byutilizing the binding ability of the antibodies with the polypeptide(I), as reagents for purifying the polypeptide (I).

The EIA or the RIA used to detect and assay the polypeptide (I) iscarried out, for example, in the following manner. The purified antibodyis fixed in an amount of 0.1 to 10 μg/well to a carrier such as a96-well plastic plate (for example, Immunoplate, Nunc, Denmark), glassbeads or plastic beads. In the case of the plastic plate or the plasticbeads, the antibody is fixed by reaction at 4° C. overnight or at roomtemperature for about 0.5 to 4 hours. In the case of the glass beads,the antibody is fixed, for example, by a method as described in Proc.Natl. Acad. Sci. U.S.A. 80, 3513-3516 (1983). Various other plates forfixation of antibodies, which are commercially available, can also beused.

A solution containing the antigen polypeptide (I) is added to the plateof the beads to which the antibody is thus fixed, followed by adsorptionreaction. The adsorption reaction is sometimes conducted at roomtemperature for about 0.2 to 2 hours, however preferably conducted atabout 4° C. overnight.

After the antigen-antibody binding reaction, an antibody to which alabeling agent is bound is added, followed by adsorption reaction. Thelabeling agents include radioisotopes, enzyme, fluorescent substancesand luminous substances. However, it is preferred to use the enzymes. Asthe enzymes, which are preferably stable and high in specific activity,there can be used peroxidases, alkaline phosphatases,β-D-galactosidases, glucose oxidases and the like. In particular,peroxidases are preferably used. Peroxidases of various origins can beused. Examples of such peroxidases include peroxidases obtained fromhorseradishes, pineapples, figs, sweet potatoes, broad beans and cone.In particular, horseradish peroxidase (HRP) extracted from horseradishesis preferable.

In binding peroxidase to the antibody, the thiol group of Fab' isutilized as the antibody molecule. For this reason, peroxidase intowhich a maleimide group is preliminarily introduced is convenientlyused.

When a maleimide group is introduced into peroxidase, a maleimide groupcan be introduced through an amino group of the peroxidase. For thispurpose, N-succinimidyl-maleimide-carboxylate derivatives can be used.N-(γ-maleimidobutyloxy) succinimide (hereinafter also referred to asGMBS for brevity) is preferably used. A certain group may thereforeintervene between the maleimide group and the peroxidase.

GMBS is reacted with peroxidase in a buffer solution having a pH of 6 to8 at about 10° to 50° C. for about 10 to 24 hours. The buffer solutionsinclude, for example, 0.1M phosphate buffer (pH 7.0). The maleimidatedperoxidase thus obtained can be purified, for example, by gelchromatography. Examples of carriers used in the gel chromatographyinclude Sephadex G-25 (Pharmacia Fine Chemical, Sweden) and Biogel P-2(Bio RAD Laboratories, U.S.A.).

The maleimidated peroxidase can be reacted with an antibody molecule ina buffer solution at about 0° to 40° C. for about 1 to 48 hours. Thebuffer solutions include, for example, 0.1M phosphate buffer (pH 6.0)containing 5 mM sodium ethylenediaminetetraacetate. Theperoxidase-labeled antibody thus obtained can be purified, for example,by gel chromatography. Examples of carriers used in the gelchromatography include Sephadex G-25 (Pharmacia Fine Chemical, Sweden)and Biogel P-2 (Bio RAD Laboratories, U.S.A.).

A thiol group may be introduced into the peroxidase to react with themaleimidated antibody molecule.

Enzymes other than the peroxidases can be directly bound to theantibodies similarly to the methods of binding the peroxidases, andknown methods which achieve such binding include, for example, theglutaraldehyde method, the periodic acid method and the water-solublecarbodiimide method.

For the enzyme-labeled antibodies, reaction substrates such as2,2'-diadino-di 3-ethylbenzothiazoline sulfonate(6)! in the case of HRPare added to develop color, thereby measuring absorbance. As to theradio-labeled antibodies, the radioactivity of the antibodies not boundto the polypeptide (I) is measured by a scintillation counter. Theabsorbance or the radioactivity of the samples are compared to thevalues to the polypeptide (I) of a known amount, whereby the polypeptide(I) can be assayed.

In addition to sandwich EIAs in which an antigen is sandwiched betweentwo kinds of antibodies and which is described later, competitive EIAsand indirect EIAs which are well known are carried out. In thecompetitive EIAs, the antibody is fixed to a carrier, and the enzyme- orradio-labeled antigen polypeptide (I) and a sample is added thereto,followed by reaction and assay of the polypeptide (I). The reaction andestimation of the labeled antigen are conducted under the conditionssimilar to those described above. In the indirect EIAs, a test sample isreacted with the antibody which is not fixed to a carrier, and theantibody which is not adsorbed is assayed with a plate to which theantigen is fixed and an anti mouse-labeled antibody. The reaction andthe measurement of the radioactivity are conducted under the conditionssimilar to those described above.

Test samples used in the assay system, such as the detection anddetermination of the polypeptide (I) of the present invention, includehumors such as urine, serum, plasma and cerebrospinal fluid, extracts ofanimal cells, and culture supernatants thereof.

As an example of the assays of the present invention, a case ishereinafter described in detail in which the peroxidase is used as thelabeling agent, but the present invention is not limited to theperoxidase.

(1) First, a test sample containing the polypeptide (I) to be assayed isadded to the antibody held on a carrier to conduct antigen-antibodyreaction, and then the conjugate of the peroxidase with the antibodyobtained above is added thereto, followed by reaction.

Test samples used in this assay system include humors such as urine,serum, plasma and cerebrospinal fluid, extracts of animal cells, andculture supernatants thereof.

The carriers on which the antibody is held in the assays of thepolypeptide (I) include, for example, gel particles such as agarose gelsfor example, Sepharose 4B and Sepharose 6B (Pharmacia Fine Chemical,Sweden)!, dextran gels for example, Sephadex G-75, Sephadex G-100 andSephadex G-200 (Pharmacia Fine Chemical, Sweden)! and polyacrylamidegels for example, Biogel P-30, Biogel P-60 and Biogel P-100 (Bio RADLaboratories, U.S.A.)!; cellulose particles such as Avicel (AsahiChemical Industry, Japan) and ion exchange cellulose (for example,diethylaminoethyl cellulose and carboxymethyl cellulose); physicaladsorbents such as glass (for example, glass balls, glass rods,aminoalkyl glass balls and aminoalkyl glass rods), silicone pieces,styrenic resins (for example, polystyrene balls and polystyreneparticles) and plates for immunoassay (for example, Nunc, Denmark); andion exchange resins such as acescent cation exchange resins for example,Amberlite IRC-50 (Rohm & Haas, U.S.A.) and Zeocurve 226 (Permutit, WestGermany)!, and alkalescent anion exchange resins (for example, AmberliteIR-4B and Dowex (Dow Chemical, U.S.A.)!.

In order to hold the antibody on the carrier, methods known in the artare applied. Examples of such methods include the cyanogen bromidemethod and the glutaraldehyde method which are described in Metabolism8, 696 (1971). As a simpler method, the antibody may be adsorbed on thesurface of the carrier.

(2) The substrate of the peroxidase is added to the reaction productobtained in (1), and then the absorbance or the fluorescent intensity ofthe resulting substance is measured, thereby knowing the enzyme activityof the above reaction product.

(3) The procedures described in (1) and (2) are preliminarily carriedout for the standard solution of the polypeptide (I) of a known amountto prepare a standard curve showing the relation between the amount ofthe polypeptide (I) and the absorbance or the fluorescent intensitythereof.

(4) The absorbance or the fluorescent intensity obtained for the testsample containing the polypeptide (I) of an unknown amount is applied tothe standard curve to determine the amount of the polypeptide (I) in thetest sample.

The above antibody can be utilized for the detection and determinationof the polypeptide (I) by Western blotting W. N. Burnette, AnalyticalBiochemistry 112, 195 (1981)!.

A specific example of Western blotting is described below.

A sample containing the polypeptide (I) is dissolved for example, insample buffer U. K. Laemmli, Nature 227, 680 (1970)!. In this case2-mercaptoethanol is either added thereto as a reducing agent (underreducing conditions) or not (under non-reducing conditions). Either ofthese conditions may be used. The resulting solution is heated at about100° C. for 5 minutes, and then subjected to electrophoresis. Anyelectrophoresis may be used as long as the protein can be separated.Specific examples of such electrophoresis include SDS-polyacrylamide gelelectrophoresis. The protein is transferred from the gel afterelectrophoresis to a nitrocellulose membrane. This method is well knownin the art, and includes, for example, the method of Burnette AnalyticalBiochemistry 112, 195 (1981)!. Then, the polypeptide (I) on thenitrocellulose membrane is detected by an immunological method. Namely,the nitrocellulose membrane is blocked, for example, with a 3% gelatinsolution, followed by a first antibody reaction. An antibody used as afirst antibody may be either antiserum or a purified antibody. However,the purified antibody is more preferable. As to the conditions of thefirst antibody reaction after blocking, any set of conditions may beapplied as long as the polypeptide (I) on the membrane can be bound tothe first antibody. For example, the first antibody reaction isconducted at room temperature for about 4 to 16 hours. After the abovefirst antibody reaction, a second antibody reaction is carried out. Asto the second antibody used, any antibody can be employed as long as itcan be bound to the first antibody and is detectable. Examples of suchantibodies include IgG coupled with a labeling enzyme. Specific examplesof labeling enzymes include horseradish peroxidase (HRP) and alkalinephosphatase. As to the conditions of the first antibody reaction, anyconditions may be applied as long as the second antibody can be bound tothe first antibody. For example, the second antibody reaction isconducted at room temperature for about 1 hour. After the above secondantibody reaction, color is developed and the band of the polypeptide(I) on the nitrocellulose membrane is detected. According to the aboveWestern blotting, the polypeptide (I) is detectable if it exists in anamount of about 50 ng or more. The polypeptide (I) in the test samplecan be determined by comparing the density of the band of the testsample to the density of the bands of a known amount of polypeptide (I).In lieu of the above first antibody, for example, the conjugate of theantibody with HRP may be used.

In order to purify the polypeptide (I), the purified antibody is coupledwith a suitable carrier such as activated agarose gel beads according toconventional methods, and packed in a column. Then, a sample containingthe polypeptide (I), such as a culture supernatant or disrupted cells,is loaded onto the antibody column to allow the sample to be adsorbedthereby, followed by washing. Then, elution is carried out with achaotropic reagent such as potassium thiocyanate (KSCN) or under suchacescent conditions that the polypeptide (I) is not inactivated. Thus,the polypeptide (I) can be efficiently purified.

The antibody column can be prepared by coupling the monoclonal antibodyof the present invention, which is, for example, purified from ascitesor other humors inoculated with the hybridoma cells, with an appropriatecarrier.

Any carrier may be used as long as the polypeptide (I) is specificallyefficiently adsorbed thereby after coupling and suitable elution isthereafter possible. By way of example, agarose gel beads in whichprimary amines of the protein are activated so as to be easily bindable,such as Affi-Gel 10 (Bio RAD), are conveniently used according to thefollowing method. The antibody is reacted with Affi-Gel 10 in a buffersolution such as a bicarbonate solution having a concentration of about0.001 to 1M, preferably about 0.1M. The reaction is conducted at about0° to 20° C. at a broad pH range for about 10 to 24 hours, preferably atabout 4° C. at a pH of about 7 to 10 for about 4 hours. With respect tothe mixing ratio of the antibody to Affi-Gel 10, the larger amount ofantibody mixed with Affi-Gel 10, the larger amount of antibody whichbecomes bound thereto. Up to about 50 mg of antibody per 1 ml ofAffi-Gel 10 can become bound. Hence, about 10 to 30 mg of the antibodyis conveniently used, considering the purification efficiency inaffinity chromatography. The antibody-carrier combined material thusformed is thoroughly washed with the buffer solution used for thereaction. Then, residual unreacted active groups are blocked by allowingthe washed material to stand for several days, by adding a compoundcontaining a primary amine such as ethanolamine-hydrochloric acid orglycine thereto to a final concentration of about 0.05 to 0.10M,followed by reaction at about 4° C. for about 1 to 4 hours, or byreacting a protein such as 1 to 5% bovine serum albumin (BSA) therewithat 4° C. overnight. The combined material thus treated is packed in anappropriate column to form the antibody column.

In purifying a sample with the above antibody column, the polypeptide(I) protein-containing sample is dissolved in a buffer solution having apH around neutrality such as phosphate buffer or Tris-hydrochloric acidbuffer, followed by adsorption by the antibody column. Then, the columnis washed with the same buffer, and then the polypeptide (I) is eluted.As eluents, the following solutions are commonly used: acetic acidsolutions, solutions containing polyethylene glycol, solutionscontaining peptides more easily bindable with the antibody than thesample, high concentration salt solutions and their combined solutions.Solutions which do not so promote the inactivation of the polypeptide(I) are preferred.

Effluents are neutralized with buffer solutions by methods known in theart. The above purification procedure can be repeated as needed.

Further, the substantially pure polypeptide (I) substantially free frompyrogens and endotoxins can be obtained by combining variouspurification techniques known in the art. The substantially purepolypeptide (I) of the present invention contains the polypeptide (I) ina concentration of 90% (w/w) or more, preferably 95% (w/w) or more.

The polypeptide (I) protein solution obtained here is subjected todialysis, and can be pulverized by lyophilization as needed. Inlyophilizing, stabilizing agents such as sorbitol, mannitol, dextrol,maltose and glycerol can be added.

When the polypeptide (I) thus obtained has sufficient activity, it isused as is. When it does not have sufficient activity, it can besufficient activated by enzymatic methods or non-enzymatic methods. Thepolypeptide (I) thus obtained shows stimulation of differentiation orproliferation of animal cells, particularly of nerve cells, and hasinducing activity for various proteins and enzymes. The polypeptide (I)is therefore expected to be used to treat nerve-related pathologicconditions such as nerve lesion.

Further, the polypeptide (I) is expected to have the same action as NGFor action similar to that of NGF.

The polypeptide (I) of the present invention is useful as reagents forstudies relating to differentiation, growth, multiplication and survivalof animal cells.

When the polypeptide (I) is used as medicine, it can be safelyadministered parenterally or orally to warm-blooded mammals (such ashumans, mice, rats, hamsters, rabbits, dogs and cats) in a powder formas is, or as pharmaceutical preparations together with pharmaceuticallyacceptable carriers, excipients or diluents.

The injections of the polypeptide (I) are prepared, for example, usingphysiological saline or aqueous solutions containing glucose or otheradjuvants by methods known in the art. The pharmaceutical preparationssuch as tablets and the capsules can also be prepared.

When the polypeptide (I) is used as the above medicines, it isadministered, for example, to the above warm-blooded animals in anappropriate amount ranging from about 1 ng/kg to 100 μg/kg daily, takinginto account the route of administration, symptoms, etc.

When the polypeptide (I) is used as the reagents for studies relating todifferentiation, growth, proliferation, activation and the like ofanimal cells, it is preferably added to a culture medium for animalcells in an amount of about 0.1 to 1,000 ng per milliliter of medium,more preferably about 1 to 100 ng per milliliter of medium. Thedifferentiation, growth, proliferation and survival of animal cells canbe assayed by incubating animal cells in the medium to which thepolypeptide (I) is added.

By using the antibodies to the polypeptide (I) or its partial peptides,the isolation, purification and assay of the polypeptide (I) can beefficiently carried out, and the application of the polypeptide (I) isachieved.

When bases, amino acids and so on are indicated by the abbreviations inthis specification and drawings, the abbreviations adopted by IUPAC-IUBCommission on Biochemical Nomenclature or commonly used in the art areemployed. For example, the following abbreviations are used. When theoptical isomer is capable of existing with respect to the amino acids,the L-form is represented unless otherwise specified.

                  TABLE 1                                                         ______________________________________                                        DNA        Deoxyribonucleic acid                                              A          Adenine                                                            C          Cytosine                                                           G          Guanine                                                            T          Thymine                                                            Ala        Alanine                                                            Arg        Arginine                                                           Asn        Asparagine                                                         Asp        Aspartic acid                                                      Cys        Cysteine                                                           Gln        Glutamine                                                          Glu        Glutamic acid                                                      Gly        Glycine                                                            His        Histidine                                                          Ile        Isoleucine                                                         Leu        Leucine                                                            Lys        Lysine                                                             Met        Methionine                                                         Phe        Phenylalanine                                                      Pro        Proline                                                            Ser        Serine                                                             Thr        Threonine                                                          Trp        Tryptophan                                                         Tyr        Tyrosine                                                           Val        Valine                                                             Boc        t-Butyloxycarbonyl                                                 MeBzl      p-Methylbenzyl                                                     Bzl        Benzyl                                                             P          Polystyrene resin for solid synthesis of peptide                   PAM        p-Oxymethylphenylacetamidomethyl resin                             AcOH       Acetic acid                                                        OBzl       Benzyl ester                                                       Tos        Tosyl                                                              Br-z       2-Bromobenzyloxycarbonyl                                           Cl-z       2-Chlorobenzyloxycarbonyl                                          ______________________________________                                    

Transformant Escherichia coli MV1184/pUNK5 obtained in Reference Example1 described below was deposited with the Institute for Fermentation,Osaka, Japan (IFO) under the accession number IFO 14832 on Feb. 10,1989. This microorganism was also deposited with the FermentationResearch Institute, Agency of Industrial Science and Technology,Ministry of International Trade and Industry, Japan (FRI) under theaccession number FERM BP-2304 on Feb. 22, 1989.

Transformant Escherichia coli BL21(DE3)/pLysS, pENGFT102 obtained inReference Example 8 described below was deposited with the Institute forFermentation, Osaka, Japan (IFO) under the accession number IFO 14903 onJul. 14, 1989. This microorganism was also deposited with theFermentation Research Institute, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, Japan (FRI)under the accession number FERM BP-2529 on Jul. 26, 1989.

Mouse N4-2 cells, mouse N46-31 cells, mouse N82-4 cells and mouseN148-62 cells obtained in Example 2(3) described below were depositedwith the Institute for Fermentation, Osaka, Japan (IFO) on Apr. 25,1990, and with the Fermentation Research Institute, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry,Japan (FRI) on May 15, 1990, under the following accession numbers.

    ______________________________________                                                      IFO   FERM BP                                                   ______________________________________                                        MoAb4-2         50241   2908                                                  MoAb46-31       50242   2909                                                  MoAb82-4        50243   2910                                                  MoAb148-62      50244   2911                                                  ______________________________________                                    

The present invention will hereinafter be described in detail with thefollowing Reference Examples and Examples. It is understood of coursethat these Reference Examples and Examples are not intended to limit thescope of the invention.

REFERENCE EXAMPLE 1 (Cloning of Polypeptide (I) cDNA)

E. coli Y1090 was infected with the human glioma-derived λgt11 cDNAlibraries (Clontech Laboratories, Inc.) and then about 6×10⁵ phages werespread on an agar plate containing NZCYM medium described in MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982),followed by cultivation at 37° C. for 5 hours. Then, a nylon membranewas placed on the plate, and removed after standing for 1 minute. Thisnylon membrane was soaked in 0.5M NaOH- 1.5M NaCl, then in 1.5MNaCl-0.5M Tris-HCl (pH 8.0), and further in 2×SSC Refer to MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982)!.After air drying, the membrane was allowed to stand at 80° C. for 2hours.

A DNA (about 0.38 kb) coding for human βNGF Nature 303, 821 (1983)! waschemically synthesized and labeled with α-³² P!dCTP by nick translation,thereby preparing a probe.

Using the nylon membrane and the probe obtained in the above,hybridization was carried out according to the method described inMolecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory(1982). Thus, the nylon membrane was soaked in a hybridization solutioncontaining the probe, and maintained at 65° C. for 16 hours. The nylonmembrane was washed with 2×SSC-0.1% SDS at room temperature, and thenwith 1×SSC-0.1% SDS at 60° C. Thereafter, positive clones were detectedby autoradiography.

A cDNA portion was cut out with EcoRI from the clone λβGN1321 thusobtained and inserted into the EcoRI site of plasmid pUCl18 (TakaraShuzo) to obtain plasmid pUNK5. Using the plasmid pUNK5 thus obtained,E. coli MV1184 (Takara Shuzo) was transformed to obtain transformant E.coli MV1184/pUNK5 (IFO 14832, FERM BP-2304).

FIG. 1 shows the restriction enzyme map of the cDNA portion includingthe polypeptide (I) cDNA contained in the plasmid pUNK5 and having awhole length of about 0.78 kb. In FIG. 1, ▭ shows an untranslatedregion, shows a propeptide code region, and shows a region coding for apolypeptide further having a threonine residue at the C-terminus of theamino acid sequence of formula 1!.

The nucleotide sequence of the cDNA portion obtained in the above wasdetermined by the dideoxy method Messing et al., Nucl. Acid. Res. 9, 309(1981)!. FIG. 2 shows the determined nucleotide sequence and the aminoacid sequence translated thereby. In FIG. 2, the region extending fromposition 1 to the N-terminus of the amino acid sequence is a portion ofthe propeptide, and the region of positions 1 to 118 and positions 1 to119 show the polypeptide having the amino acid sequence of formula 1!and the polypeptide further having a threonine residue at the C-terminusof the amino acid sequence of formula 1!, respectively.

FIG. 3 shows the amino acid sequence of the polypeptide (I) determinedby the above method, in comparison with the amino acid sequence of thehuman βNGF described in Ullrich et al., Nature 303, 821 (1983). In FIG.3, the upper row indicates the sequence of 119 amino acids of thepolypeptide (I), and the lower row indicates the amino acid sequence ofthe human βNGF. The same amino acid residue portions are boxed In thefigure, "-" only shows a chemical bond.

As apparent from this comparison, the sequence of 119 amino acids of thepolypeptide (I) of the present invention has a homology of about 60%with the amino acid sequence of the above human βNGF.

Further, when the sequence of 119 amino acids of the polypeptide (I)determined as described above is compared with the amino acid sequenceof the mouse βNGF shown in Angeletti et al., Proceedings of NationalAcademy of Sciences, U.S.A. 68, 2417 (1971) and Scott et al., Nature302, 538 (1983), it has a homology of about 60%.

REFERENCE EXAMPLE 2 (Recloning of Polypeptide (I) cDNA)

Using the EcoRI-AhaIII fragment containing the 5'-terminal side of thepolypeptide (I) cDNA portion contained in the pUNK5 obtained inReference Example 1 as a probe, one of the human glioma-derived cDNAlibraries (Clontech Laboratories, Inc.) was cloned in a manner similarto that of Example 1. A cDNA portion was cut out with EcoRI from one ofmany positive clones, λHNT31, thus obtained, was inserted into the EcoRIsite of plasmid pUCl19 (Takara Shuzo) to obtain plasmid pHNT2. FIG. 4shows the restriction enzyme map of a polypeptide (I) cDNA (about 1.1kb) inserted into the plasmid pHNT2. In FIG. 4, shows a signal peptidecode region, shows a propeptide code region, and shows a region codingfor a polypeptide further having a threonine residue at the C-terminusof the amino acid sequence of formula 1!.

The nucleotide sequence of the cDNA portion obtained in the above wasdetermined by the dideoxy method. FIG. 5 shows the determined nucleotidesequence and the amino acid sequence translated thereby. In FIG. 5,"Signal" indicates the signal peptide, "Pro" indicates the propeptideand "Mature" indicates the polypeptide (I) (mature protein).

REFERENCE EXAMPLE 3 (Construction of Polypeptide (I) Expression Vectorfor Escherichia coli)

The polypeptide (I) cDNA inserted into the plasmid pUNK5 obtained inReference Example 1 has an ScaI site near the region coding for the 11thtyrosine residue from the N-terminus of the polypeptide (I), and an NsiIsite downstream from a terminating codon of the polypeptide (I) by 50bases (refer to FIGS. 2, 4 and 5). A 0.3-kb ScaI-NsiI segment wasisolated from the plasmid pUNK5, and adapters NGFTE-1 (35mer), NGFTE-2(33mer), NGFTE-3 (7mer) and NGFTE-4 (15mer) were ligated thereto with T4DNA ligase, followed by treatment with restriction enzymes NdeI andBamHI. Thus, a 0.3-kb NdeI-BamHI fragment was obtained (refer to FIG.6).

These adapters are as follows: ##STR3##

The expression vector pET-3C having a T7 promoter Rosenberg et al., Gene56, 125 (1987)! was similarly cleaved with NdeI and BamHI to isolate a4.4-kb NdeI-BamHI fragment.

The 4.4-kb NdeI-BanHI fragment obtained above was ligated to the 0.3-kbNdeI-BamHI fragment with T4 DNA ligase, and then the ligated fragmentwas inserted into E. coli DH1 to prepare a transformant. A plasmidisolated from the resulting ampicillin-resistant transformant strain E.coli DH1/pENGFT102! was named pENGFT102 (FIG. 6).

REFERENCE EXAMPLE 4 (Preparation and Expression of Transformant)

Using the polypeptide (I) expression vector pENGFT102 obtained inReference Example 3, E. coli BL21(DE3) Gene 56, 125 (1987)! wastransformed to obtain transformant E. coli BL21(DE3)/pENGFT102.

The transformant E. coli BL21(DE3)/pENGFT102 was cultivated on 5 ml ofLB culture medium containing 50 μg/ml ampicillin and 0.2% glucose in atest tube at 37° C. for 16 hours. 1 ml of the resulting culture wastransferred into a 200-ml flask containing 20 ml of the same medium, andcultivated at 37° C. When the Klett value reached 170 to 200, IPTG wasadded thereto to give a final concentration of 0.4 mM, and thecultivation was further continued for 3 hours. Cells collected from 30μl of the resulting culture were suspended in 30 μl of sample buffer 50mM Tris-HCl (pH 6.8), 2 mM EDTA, 1% SDS, 1% mercaptoethanol, 8%glycerol, 0.025% Bromophenol Blue1, and heated for 5 minutes, followedby electrophoresis on 16% polyacrylamide gels containing 0.1% SDS. Afterelectrophoresis, the gels were dyed with Coomassie Brilliant Blue. As aresult, a 15-kilodalton (kDa) protein which was not detected in E. coliBL21(DE3)/pET-3C obtained by transforming E. coli BL21(DE3) by the abovevector pET-3C, was detected in E. coli BL21(DE3)/pENGFT102. The amountof the 15-kDa protein produced was about 10% of the total proteins. Thisprotein was also detected by the Western blotting method using rabbitanti-mouse NGF antibody (Collaborative Research, Inc. U.S.A.).

REFERENCE EXAMPLE 5 (Construction of Polypeptide (I) Expression Vectorfor Animal Cells)

A 1.1-kb EcoRI fragment containing the polypeptide (I) cDNA was isolatedfrom the plasmid pHNT2 obtained in Reference Example 2. Expressionvector pTB389 (described in Japanese Patent Unexamined Publication(Laid-open) No. 64-2572/1989 corresponding to EP-251,244A) was similarlycleaved with EcoRI. The resulting fragment was ligated to the above1.1-kb EcoRI fragment containing the polypeptide (I) cDNA with T4 DNAligase, and then the ligation mixture was used for the transformation ofE. coli DH1 (Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, p.505, 1982). A plasmid was isolated from the resultingampicillin-resistant transformant E. coli DH1/pNTK26!, and this plasmidwas named pNTK26.

A 1.1-kb ClaI-HindIII fragment containing an Abelson Mouse leukemiavirus (A-MuLV) LTR region was isolated from plasmid pTB505 (described inJapanese Patent Unexamined Publication (Laid-open) No. 62-175182/1987corresponding to EP-225,701A). The plasmid pNTK26 was similarly cleavedwith restriction enzymes ClaI and HindIII, and the smaller fragment wasremoved. Then, the resulting fragment was ligated to the above 1.1-kbClaI-HindIII fragment containing the A-MuLV LTR region with T4 DNAligase, and the ligation mixture was used for the transformation of E.coli DH1 to give ampicillin-resistant transformant E. coli DH1/pNTL145.Plasmid pNTL145 was isolated from the transformant thus obtained (FIG.7).

REFERENCE EXAMPLE 6 (Construction of Polypeptide (I) Expression Vectorfor Animal Cells)

A 0.86-kb EcoRI-AhaIII fragment containing the regions coding for thesignal peptide, the propeptide and the polypeptide (I) in thepolypeptide (I) cDNA was isolated from the plasmid pHNT2 obtained inreference Example 2 (as to the location of the AhaIII site, refer toFIGS. 4 and 5). The 5'-terminus (EcoRI) of the resulting fragment wasmade flush with Klenow fragment, and then a XhoI linker pCCTCGAGG wasligated to each terminus thereof with T4 ligase, followed by treatmentwith XhoI. Thus, a 0.86-kb XhoI fragment was obtained.

The expression vector pKSV-10 (Pharmacia) for animal cells was cleavedwith restriction enzyme BglII, and then both ends (XhoI) of theresulting fragment was made flush with Klenow fragment. The XhoI linker(previously described) was added thereto, and this fragment was ligatedto the above 0.86-kb XhoI fragment with T4 DNA ligase. The ligatedfragment was used to transform E. coli DH1. Plasmid pNTS101 was isolatedfrom the resulting ampicillin-resistant transformant E. coli DH1/pNTS101(FIG. 8).

REFERENCE EXAMPLE 7 (Expression of Polypeptide (I) cDNA in Animal Cells)

Monkey COS-7 cells were cultivated in monolayer in Dulbecco's modifiedEagle's medium (DMEM medium) (Flow Laboratories) containing 10% fetalcalf serum, followed by exchanging the medium for the same medium. After4 hours from the exchange, calcium phosphate gels containing theexpression vector pTB389, 10 μg of the polypeptide (I) expression vectorpNTK26 and the polypeptide (I) expression vector pNTL145, respectively,were prepared according to the method known in the art Graham et al.,Virology 52, 456 (1973)!, and added to cells to obtain transformantsCOS-7/pTB389, COS-7/pNTK26 and COS-7/pNTL145, respectively. These cellswere cultivated in a carbon dioxide incubator for 4 hours, and thentreated with glycerol Gorman et al., Science 221, 551 (1983)!, followedby cultivation for 3 days. Cultures after cultivation were centrifugedto obtain culture supernatants. PC12 cells were cultivated in thepresence of the respective supernatants according to the methoddescribed in Brain Research 133, 350 (1977) and Experimental CellResearch 145, 179 (1983), and the proportions of cells whose neutritesoutgrew to at least twice the diameter of the cells were calculated. Theresults are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                              Proportion of Cells                                                 Culture   with Neurites Twice                                                 Supernatant                                                                             the Size of Cell                                        Vector      (μl)   Diameter (%)                                            ______________________________________                                        pTB389      40        11                                                      pNTK26      40        17                                                      pNTL145     40        20                                                      ______________________________________                                    

Using a culture supernatant obtained by a method similar to thatdescribed above, the effect on acetylcholine (ACh) content ofco-cultured septal and basal forebrain neurons M. Kakihana and M. Suno,Nerve Chemistry 27, 166 (1988)! was investigated.

Septum and basal forebrain were dissected from 17-day fetal rat brains,and nerve cells were isolated therefrom in accordance with the method ofHatanaka et al. Develop. Brain Res. 30, 47 (1986)!. The cells wereseeded on a 48-well plate pretreated with 100 μg/ml of poly-L-ornithineat a density of about 1×10⁶ cells/cm² /well, and cultivated in 500 μl ofa serum-free DME/F12/N2 medium for 24 hours. After removing the mediumby suction, 500 μl of DME/F12/10% FCS and the supernatant of thespecimen were added. After 2 days, the medium was exchanged for 750 μlof the same medium, and the supernatant was added again, followed bycultivation for 2 days. The supernatant was added sequentially. Namely,50 μl of the supernatant was added for the former two days and 75 μlthereof for the latter two days to give a final concentration of 10%.When mouse NGF (7S-NGF) purchased from Wako Pure Chemical Industries wasused, it was diluted with 0.1% ovalbumin/PBS, and 10 μl thereof wasadded.

After 4 days from the addition of the supernatant, the medium wasremoved by suction, and 500 μl of cooled 0.3N PCA and 20 to 60 pmoles/20μl of ethylhomocholine (EHC) were added thereto for measurement of ACh.After gentle stirring, 500 μl of the solution was transferred to anEppendolf microtube. Subsequent operations were carried out inaccordance with the previously reported methods, and the amount of AChwas measured by use of a high-performance liquidchromatography/electrochemical detector (HPLC/ECD) system. Afterextraction of ACh, the cells were dissolved in 500 μl of 1N NaOH, andthe amount of protein was assayed (Bio-RAD protein assay). Dunnett'st-test was used for statistical treatment.

The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                                         Amount of                                                           Number    Acetylcholine                                Experiment                                                                             Sample        of Wells  (pmol/mg protein)                            ______________________________________                                        1        Mouse NGF 0 ng/ml                                                                           6         492 ± 31                                           Mouse NGF 0.1 ng/ml                                                                         6         526 ± 14                                           Mouse NGF 1 ng/ml                                                                           6         600 ± 31                                           Mouse NGF 10 ng/ml                                                                          6         775 ± 29                                           Supernatant (10%) of                                                                        6         582 ± 22                                           COS-7/pTB 389                                                                 Supernatant (10%) of                                                                        6         652 ± 13                                           COS-7/pNTL 145                                                       2        Supernatant (10%) of                                                                        4         332 ± 7                                            COS-7/pTB389                                                                  Supernatant (10%) of                                                                        6         395 ± 7                                            COS-7/pNTL 145                                                       ______________________________________                                    

REFERENCE EXAMPLE 8 (Production of Polypeptide (I) by Escherichia coli)

Escherichia coli BL21(DE3) Gene 56, 125 (1987)! was transformed by thepolypeptide (I) expression vector pENGFT102 obtained in ReferenceExample 3 and T7 lysozyme expression vector pLysS to obtain transformantEscherichia coli BL21(DE3)/pLysS, pENGFT102 (IFO 14903, FERM BP-2529).

The transformant E. coli BL21(DE3)/pLysS, pENGFT102 was cultivated in LBmedium 1% tryptone (Difco), 0.5% yeast extract, 0.5% NaCl! containing 50μg/ml of ampicillin, 10 μg/ml of chloramphenicol and 0.2% glucose at 37°C. for 16 hours with shaking. The culture (12.5 ml) was transferred intoa 1-liter Erlenmeyer flask containing 250 ml of the same medium, andcultivated at 30° C. for 3 hours with shaking. Thereupon, the Klettvalue of the culture reached 170. Isopropyl-β-D(-)-thiogalactopyronosidewas added to this culture at a final concentration of 0.1 mM, and theresulting solution was cultivated at 30° C. for 3 hours with shaking.Cells collected from 30 μl of the culture solution thus obtained weresuspended in 30 μl of sample buffer Laemmli, Nature 227, 680 (1970)!,and heated at 100° C. for 5 minutes, followed by electrophoresis on 16%polyacrylamide gels containing 0.1% SDS. The proteins on the gels weretransferred to a nitrocellulose membrane according to the method ofBurnette Analytical Biochemistry 112, 195 (1981)!, and then, Westernblotting was carried out using the rabbit anti-mouse NGF antibody(Collaborative Research Inc. U.S.A.) and the affinity-purifiedHRP-linked goat anti-rabbit IgG (Bio RAD, U.S.A.). As a result, thepolypeptide (I) having a molecular weight of 15 kilodaltons (kDa) wasdetected.

When gels obtained in a manner similar to that described above andsubjected to electrophoresis were dyed with Coomassie Brilliant Blue, a15-kDa protein corresponding to the polypeptide (I) was detected, andthe amount of the 15-kDa protein produced was estimated to be about 10%of the total proteins.

REFERENCE EXAMPLE 9 (Isolation of Polypeptide (I))

The culture (3.75 liter) of the transformant E. coli BL21(DE3)/pLysS,pENGFT102 obtained in Reference Example 8 was centrifuged to give 17 g(wet) of cells. The cells were suspended in 375 ml of 50 mM Tris-HCl (pH8.0) and freeze-thawed, followed by treatment with a sonic oscillator(Kaijo Denki, 2A, 2 minutes) 3 times. The broken cell suspension wascentrifuged, and the resulting precipitate was dissolved in 60 ml of 5Mguanidine hydrochloride-5 mM EDTA-1 mM PMSF-0.1 mM APMSF-20 mMdithiothreitol (DTT)-50 mM sodium phosphate buffer (pH 6.0). Thesolution thus obtained was applied to a Sephacryl S-200 columnequilibrated with 2M guanidine hydrochloride-5 mM EDTA-0.1 mM APMSF-5 mMDTT-25 mM sodium phosphate buffer (pH 6.0), and the fractions in whichthe polypeptide (I) was detected by the Western blotting method(previously described) were collected (volume=300 ml). This solution wasconcentrated by use of an ultrafilter equipped with a YM5 membrane(Amicon), and 50 ml of the resulting concentrated solution was appliedto the Sephacryl S-200 column as described above. Thus, 164 ml of asolution containing 328 mg of the purified polypeptide (I) was obtained.The purity was investigated by SDS-polyacrylamide gel electrophoresis.As a result, it was confirmed that the resulting purified polypeptide(I) was substantially homogeneous.

A solution containing the above purified polypeptide (I) was loaded ontoan Ultrapore RPSC column (0.46×7.5 cm, Altex), and chromatographed byhigh-performance liquid chromatography (HPLC) with a trifluoroaceticacid-acetonitrile eluent solvent system to obtain the homogeneouspolypeptide (I). The N-terminal amino acid sequence of the resultingpolypeptide (I) was determined with a gas phase protein sequencer (Model470A, Applied Biosystems). Consequently, the N-terminal amino acidsequence of the purified polypeptide (I) agreed with the N-terminalamino acid sequence of the polypeptide (I) deduced from the nucleotidesequence of cDNA except that a methionine residue was added to theN-terminus (Table 4).

                                      TABLE 4                                     __________________________________________________________________________    N-terminal Amino Acid Sequence                                                         1  2  3  4  5  6  7  8  9  10                                        __________________________________________________________________________    Sequence Deter-                                                                        Met                                                                              Tyr                                                                              Ala                                                                              Glu                                                                              His                                                                              Lys                                                                              Ser                                                                              His                                                                              Arg                                                                              Gly                                       mined from                                                                    Purified Sample                                                               Sequence Deduced                                                                       Tyr                                                                              Ala                                                                              Glu                                                                              His                                                                              Lys                                                                              Ser                                                                              His                                                                              Arg                                                                              Gly                                                                              Glu                                       from cDNA                                                                     __________________________________________________________________________

The amino acid composition of the purified sample obtained above wasdetermined by the ninhydrin method. The results obtained show that, theobserved values substantially agreed with the theoretical valuescalculated from the polypeptide (I) to the N-terminus of which amethionine residue was added (Table 5).

                  TABLE 5                                                         ______________________________________                                        Amino Acid Composition                                                                  Experimental.sup.1)                                                                    Theoretical.sup.2)                                                   Value    Value                                                      ______________________________________                                        Asp         10.3       11                                                     Thr         8.3        9                                                      Ser         10.0       12                                                     Glu         11.0       11                                                     Pro         1.8        2                                                      Gly         7.9        8                                                      Ala         5.1        5                                                      Cys         5.9        6                                                      Val         8.4        9                                                      Met         1.0        1                                                      Ile         6.8        7                                                      Leu         5.1        5                                                      Tyr         5.2        5                                                      Phe         1.1        1                                                      Lys         9.6        10                                                     His         3.6        4                                                      Arg         9.3        10                                                     Trp         3.6        4                                                      ______________________________________                                         .sup.1) Calculated taking Glu as 11.                                          .sup.2) Calculated with a methionine residue was added to the Nterminus o     the polypeptide (I).                                                     

A solution (protein concentration: 2 mg/ml) containing the abovepurified polypeptide (I) was diluted with 2M guanidine hydrochloride-5mM EDTA-0.1 mM APMSF-5 mM DTT-25 mM sodium phosphate buffer (pH 6.0) soas to give a protein concentration of 10 μg/ml. The diluted solution wasdialyzed against a 50-fold amount of 1 mM EDTA-50 mM NaHCO₃ -Na₂ CO₃ (pH10.0) at 4° C. for 16 hours and further dialyzed against the freshlyintroduced same buffer for 4 hours. The effect of the resulting dialyzedfluid on PC12 cells was examined in accordance with the method describedin Brain Research 133. 350 (1979) and Experimental Cell Research 145,179 (1983). As a result, it was observed that 6% of the PC12 cells hadneurites by addition of the inner dialyzed fluid, and 2% thereof hadneurites having a length of at least 2 times the diameter of the cellbody. On the other hand, with 1 mM EDTA-50 mM NaHCO₃ -Na₂ CO₃ (pH 10.0)as a control, not more than 0.5% of the cells had neurites.

EXAMPLE 1 (Immunization)

BALB/c mice (female, 8 weeks old) were intradermally injected with 10 μgof the antigen polypeptide (I) (obtained in Reference Example 9) whichwas dissolved in 0.4 ml of Freund's complete adjuvant (Difco). Threeweeks later, the mice were intradermally given again 10 μg of theantigen polypeptide (I) dissolved in 0.4 ml of Freund's completeadjuvant. 3 weeks later, a similar additional immunization was carriedout. Two weeks after the additional immunization, the mice wereintravenously inoculated with 10 μg of the polypeptide (I) dissolved inphysiological saline.

EXAMPLE 2

(1) Cell Fusion

Three days after the final inoculation, the spleens were removed fromthe mice immunized in Example 1 to obtain cells to be used for cellfusion. These cells were suspended in a medium prepared by mixing Iscovemedium with Ham F-12 medium in a ratio of 1:1. This mixed medium ishereinafter referred to as IH medium.

Mouse myeloma cells P3-X63-Ag·SUI were subcultured in RPMI 1640 mediumcontaining 10% fetal calf serum under an atmosphere of 5% carbon dioxideand 95% air.

Cell fusion was carried out in accordance with the method established byKohler and Milstein G. Kohler and C. Milstein, Nature 256, 495 (1975)!.The above myeloma cells (2.9×10⁷ cells) were mixed with the immunizedlymphocytes (5×10⁸ cells) obtained by the above method, and the mixturewas centrifuged. Then, 0.3 ml of 45% polyethylene glycol 6000(hereinafter referred to as PEG 6000) dissolved in IH medium wasdropwise added thereto. The PEG 6000 solution was preheated to 37° C.and slowly added. After 5 minutes, the IH medium preheated to 37° C. wasadded thereto at a rate of 0.5 ml/min until the total volume was 10 ml.The solution was then centrifuged at room temperature at 600 rpm for 15minutes to remove a supernatant. The resulting cell precipitate wassuspended in 200 ml of IH medium containing 20% calf serum. Thesuspension was seeded in a 96-well microtiter plate (Nunc) in an amountof 200 μl/well. One day later, IH medium (containing 20% calf serum)supplemented with HAT (1×10⁻⁴ M hypoxanthine, 4×10⁻⁷ M aminopterin,1.6×10⁻⁵ M thymidine) was added to the microtiter plate in an amount of100 μl/well. The IH medium supplemented with HAT is hereinafter referredto as HAT medium. Further every 3 days, one-half the amount of themedium was exchanged for HAT medium. The cells which thus grew werehybrid cells.

(2) Screening for Antibody-Producing Cells

Preliminarily, a hybrid cell culture supernatant was added in an amountof 100 μl/well to a 96-well polystyrene microtiter plate to which thepolypeptide (I) had been fixed, and incubated at 37° C. for 1 hour.After removal of the resulting supernatant and washing, the horseradishperoxidase (HRP)-labeled anti-mouse IgG goat antibody (Kappel) was addedas the second antibody, and incubated at 37° C. for 1 hour. After thesecond antibody was removed and the wells were thoroughly washed,coloring reaction was conducted by adding a reaction substrate (EIAmethod). By this method, the high antibody titer was observed in 4wells.

(3) Cloning of Hybrid Cells

The cells in these wells were spread to 0.5 cell per well on a 96-wellmicrotiter plate on which 10⁴ cells/well of mouse thymocytes hadpreliminarily been spread as vegetative cells, and cloning was carriedout. As a result, there were obtained 4 clones, MoAb4-2 cells (IFO50241, FERM BP-2908), MoAb46-31 cells (IFO 50242, FERM BP-2909),MoAb82-4 cells (IFO 50243, FERM BP-2910) and MoAb148-62 cells (IFO50244, FERM BP-2911). The deposits FERM BP-2908, FERM BP-2909, FERMBP-2910 and FERM BP-2911 were each made on May 15, 1990 under theBudapest Treaty at Fermentation Research Institute Agency of IndustrialScience and Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken305, Japan.

Table 6 shows the results of measurement of the antibody titer in thesupernatants of these cells.

                  TABLE 6                                                         ______________________________________                                        Culture supernatant                                                                                             Parent Polypeptide                                                            strain (1)                                        MoAb    MoAb    MoAb  Moab  myeloma                                                                              immunized                            Dilution                                                                            4-2     46-31   82-4  148-62                                                                              cell   mouse serum                          ______________________________________                                        X 80  2.48    2.42    0.61  1.51  0.02   2.47                                 X 160 2.35    2.14    0.42  1.37  0.02   2.42                                 X 1280                                                                              0.81    0.58    0.11  0.65  0.01   0.99                                 X 2560                                                                              0.49    0.34    0.07  0.43  0.01   0.57                                 ______________________________________                                    

In Table 6, the values show the absorption at a wavelength of 492 nm.

The cloned cells were stored in liquid nitrogen, adding dimethylsulfoxide to IH medium containing 20% calf serum to a concentration of10%.

EXAMPLE 3 (Immunoglobulin Class of Monoclonal Antibodies)

The mouse antibodies obtained in Example 2 were reacted with variousimmunoglobulin samples by a subclass detecting kit (Bio RAD). Theresults are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Immuno-                                                                       globulin                                                                             Monoclonal antibody of this invention                                  sample MoAb4-2   MoAb46-31 MoAb82-4 MoAb148-62                                ______________________________________                                        IgG1   -         -         +        +                                         IgG2a  -         -         -        -                                         IgG2b  +         +         -        -                                         IgG3   -         -         -        -                                         IgM    -         -         -        -                                         IgA    -         -         -        -                                         ______________________________________                                    

In Table 7, "+" indicates that the reaction is positive, and "-"indicates that the reaction is negative.

Table 7 shows that the antibodies produced by MoAb4-2 and MoAb46-31belong to IgG2b in immunoglobulin and that the antibodies produced byMoAb82-4 and MoAb148-62 belong to IgG1 in immunoglobulin.

EXAMPLE 4 (Preparation of Ascites Containing Monoclonal Antibodies)

For each of the hybridomas MoAb4-2, MoAb46-31, MoAb82-4 and MoAb148-62,2×10⁶ cells were injected into the mice preliminarily givenintraperitoneally 0.5 ml of mineral oil. After 10 days, 2 to 4 ml/mouseof ascites was collected. From the respective hybridomas were obtainedmonoclonal antibodies MoAb4-2, MoAb46-31, MoAb82-4 and MoAb148-62.

EXAMPLE 5 (Purification of Antibodies from Ascites)

According to the method described in Example 4, each of the monoclonalantibodies MoAb4-2, MoAb46-31, MoAb82-4 and MoAb148-62 were injectedinto 5 mice to obtain 20 to 30 ml of ascites. The ascites wascentrifuged at 2,000 rpm for 10 minutes in a centrifuge (Hitachi, Japan)to remove cells, and then centrifuged at 22,000 rpm for 2 hours at 4° C.in a Spinco SW28 rotor (Beckman, U.S.A.) to remove insoluble proteins,fats and the like. Ammonium sulfate was added to the supernatant to asaturated concentration of 40%, followed by gently stirring in ice for 1hour. The precipitate was centrifuged at 4° C. at 15,000 rpm for 30minutes by a Serval SS34 rotor (du Pont, U.S.A.). After recovery, theprecipitate was dissolved in 10 mM potassium phosphate buffer (pH 6.8),and purified using a hydroxyapatite column (HCA-column). In this case,10 mM potassium phosphate buffer (pH 6.8) was used as an initiationbuffer, and 500 mM sodium phosphate buffer was used as an elutionbuffer. Elution was effected by a linear gradient from the initiationbuffer to the elution buffer. The eluted antibodies were stored at 4° C.

EXAMPLE 6 (Preparation of HRP-Labeled Antibody)

(1) The monoclonal antibody MoAb4-2 obtained in Example 5 wasconcentrated to 2 mg/ml or more, and then dialyzed against 0.2 Mphosphate buffer (pH 7.0). An N,N'-dimethylformamide (DMF) solution ofS-acetylmercaptosuccinic anhydride (Aldrich, U.S.A.) was added in anamount of 50 μl to 0.7 ml of 4.9 mg/ml monoclonal antibody MoAb4-2 togive a concentration of 11.5 mg/ml. The air in a reaction vessel wasreplaced with nitrogen gas. The vessel was sealed, followed by stirringat room temperature for 1 hour to introduce an SH group or SH groupsinto the monoclonal antibody. Unreacted S-acetylmercaptosuccinicanhydride was inactivated by treatment with 130 μl of 0.2M Tris-HCl (pH7.0), 13 μl of 0.2M EDTA and 130 μl of 2M hydroxylamine (pH 7.0) at roomtemperature for 10 minutes. The monoclonal antibody MoAb4-2 wassubjected to gel filtration using a Sephadex G-25 column (1 cmdiameter×80 cm, Pharmacia, Sweden) (flow rate: 20 ml/hour).

(2) In 1.4 ml of 0.1M phosphate buffer (pH 6.8) was dissolved 10 mg ofhorseradish peroxidase (HRP, Grade I, Behringer Manheim, West Germany).Concurrently, 14 mg of N-hydroxysuccinimide ester ofN-(4-carboxycyclohexylmethyl)maleimide was dissolved in 335 μl of DMF,and 100 μl of the resulting solution was added to the HRP solution. Theair in a reaction vessel was replaced with nitrogen gas. The vessel wassealed, followed by stirring at room temperature for 1 hour. Then, themaleimide group-introduced HRP fractions were separated by gelfiltration using the Sephadex G-25 column (previously described).

(3) 6 ml of the SH group-introduced antibody MoAb4-2 fractions obtainedin (1) and 2 ml of the maleimide group-introduced HRP fractions obtainedin (2) were mixed with each other. The mixture was concentrated to 1 mlunder reduced pressure using a collodion bag (Sartorius, West Germany),followed by reaction at 4° C. for 20 hours. After the reaction, theHRP-introduced antibody was applied on an Ultrogel AcA44 column (1 cmdiameter×80 cm, LKB, Sweden) for fractionation (flow rate: 10 ml/hour).Of the eluted peak fractions, the fractions having the highest HRPnumber per molecule of antibody showed 2.4 HRP/antibody.

EXAMPLE 7 (Assay of Polypeptide (I) by EIA Using Monoclonal Antibody)

The monoclonal antibody MoAb82-4 obtained in Example 5 was diluted to 10μg/ml with carbonate buffer (pH 9.6), and the above diluted antibody waspoured in an amount of 100 μl/well into an immunoplate (Nunc, Denmark),followed by standing at 4° C. overnight to adsorb the monoclonalantibody to the plate. After removing the antibody not reacted, theplate was washed with PBS 3 times, and PBS containing 0.01% merthiolateand 5% bovine serum albumin (BSA) was added thereto in an amount of 300μl/well, followed by standing at 4° C. overnight.

The polypeptide (I) obtained in Reference Example 9 was diluted with PBScontaining 1% BSA. Concurrently, the BSA solution was removed from theplate obtained above, and the plate was washed with PBS 5 times. Then,the above diluted polypeptide (I) was added thereto in an amount of 100μl/well to adsorb the polypeptide (I) to the plate at 4° C. overnight.After removing the polypeptide (I) not reacted, the plate was washedwith PBS 5 times. The HRP- linked antibody (HRP-MoAb4-2) obtained inExample 6 was diluted 1/1,000 with 1% BSA, and the diluted antibodysolution was added in an amount of 100 μl/well to the plate, followed byreaction at room temperature for 2 hours. After removing the antibodysolution, the plate was washed 10 times with PBS, and a peroxidasesubstrate (Sigma) was added in an amount of 100 μl/well thereto todevelop color for comparative assay.

FIG. 9 shows a detection curve of the polypeptide (I). In this graph,the concentration of the polypeptide (I) added is plotted as abscissaand the absorbance of the color developed with HRP-MoAb4-2 as ordinate.This graph reveals that the polypeptide (I) having a concentration of0.5 ng/ml can be detected.

EXAMPLE 8 (Determination of Antigen Recognition Sites)

The antigen recognition sites of the 4 antibodies purified in Example 4were examined by competitive binding inhibition experiments. Ascompetitive peptides, there were used synthetic peptides pep1:Tyr-Ala-Glu-His-Lys-Ser-His-Arg-Gly-Glu-Tyr-Ser-Val-Cys and pep2:Cys-Ala-Leu-Ser-Arg-Lys-Ile-Gly-Arg.

The synthetic peptides were diluted with a PBS solution containing 5%BSA to a concentration of 100 μg/ml or 0.39 μg/ml. For the purifiedmonoclonal antibodies obtained in Example 4, MoAb4-2 and MoAb46-31 werediluted to 0.05 μg/ml, MoAb82-4 was diluted to 12.5 μg/ml, andMoAb148-62 was diluted to 0.78 μg/ml so that the amount of antibodyshowed 0.7 to 1.0 at an absorbance of 493 nm. As a diluent, the PBSsolution containing 5% BSA was used. The diluted competitive peptide wasadded to the diluted antibody solution. After stirring, the mixture wasmaintained at 37° C. for 30 minutes.

The amount of antibody not bound in this solution was assayed by the HIAshown in Example 7.

The results obtained when the synthetic peptides were used are shown inFIGS. 10 to 13. FIGS. 10 to 13 show the results for the monoclonalantibodies MoAb4-2, MoAb46-31, MoAb82-4 and MoAb148-62, respectively.Referring to these figures, open circles (--o--) when pep1 was used, andclosed circles (--•--) when pep2 was used. In these figures, theabsorbance (at a wavelength of 493 nm) of a color coupler is plotted asordinate.

FIGS. 10 and 11 show that the monoclonal antibodies MoAb4-2 andMoAb46-31 recognized the N-terminal 1st to 14th amino acid region of thepolypeptide (I).

FIGS. 12 and 13 show that the monoclonal antibodies MoAb82-4 andMoAb148-62 did not suffer from the inhibition by these syntheticpeptides. This reveals that the monoclonal antibodies MoAb82-4 andMoAb148-62 recognized amino acids other than the N-terminal 1st to 14thamino acid region and the C-terminal 1st to 9th region.

From the above, the recognition sites of the 4 monoclonal antibodies aresummarized as shown in Table 8.

                  TABLE 8                                                         ______________________________________                                                Monoclonal antibody                                                           4-2 46-31       82-4   148-62                                         ______________________________________                                        pep1      +     +           -    -                                            pep2      -     -           -    -                                            ______________________________________                                    

In Table 8, "+" indicates that the competitive hindrance occurred, and"-" indicates that the competitive hindrance did not occur.

EXAMPLE 9 (Detection of Human Polypeptide (I) by Western Blotting)

The polypeptide (I) obtained according to the method described inReference Example 9 was subjected to 15% acrylamide gel electrophoresisU. K. Laemmli, Nature 227, 680-685 (1970)!, and then transferred to anitrocellulose membrane using Sartblot (Sartorius) J. Kyshse-Anderson,Journal of Biochemical and Biophysical Methods 10, 203-209 (1984)!. Thismembrane was washed with TBS 20 mM Tris-HCl (pH 7.5), 0.5M NaCl! twicefor 5 minutes at a time, and then allowed to stand at room temperaturefor 1 hour in TBS containing 5% BSA to block unreacted portions on themembrane. The membrane was then washed with TBS containing 0.05% Tween20 (TTBS) 3 times for 5 minutes at a time. The monoclonal antibodyMoAb4-2, MoAb46-31, MoAb82-4 or MoAb148-62 obtained in Example 4 wasdiluted 1,000 times with TTBS containing 5% BSA. in which the abovenitrocellulose membrane was immersed. Then, the reaction was conductedat room temperature for 1 hour. The reaction solution was removed andthe membrane was washed with TTBS 3 times for 5 minutes at a time. Then,a second antibody, alkaline phosphatase-labeled anti-mouse IgG goatserum (Bio RAD, U.S.A.), diluted 2,000 times with TTBS containing 5% BSAwas added thereto, followed by reaction at room temperature for 1 hour.The membrane was washed with TTBS 3 times for 5 minutes at a time andfurther with TBS 2 times for 5 minutes at a time. Then, the membrane wasreacted at room temperature for 15 minutes with 66 μl of NBT (nitro bluetetrazolum, 50 mg/ml in 70% dimethylformamide) and 33 μl of BCIP(5-bromo-4-chloro-3- indolyl phosphate, 50 mg/ml in dimethylformamide)(Promega, U.S.A.) dissolved in AP buffer 100 mM Tris-HCl (pH 9.5), 100mM NaCl, 5 mM MgCl₂ ! as color-producing agents. FIG. 15 shows theresults of Western blotting when the monoclonal antibody MoAb4-2 wasused as the first antibody. 500 ng of the polypeptide (I) waselectrophoresed and transferred in lane 1, 250 ng of the polypeptide (I)in lane 2, 125 ng of the polypeptide (I) in lane 3 and 62.5 ng of thepolypeptide (I) in lane 4. "M" represents a marker, and the figures onthe left side represents its molecular weight.

When MoAb46-31, MoAb82-4 and MoAb148-62 were used in lieu of the aboveMoAb4-2, the polypeptide (I) was also detected.

EXAMPLE 10 (Preparation of Anti-N-Terminal Peptide Antibody)

(1) Synthesis of N-Terminal Peptide

Synthesis ofH-Tyr-Ala-Glu-His-Lys-Ser-His-Arg-Gly-Glu-Tyr-Ser-Val-Cys-OH

This peptide was synthesized by a solid-phase synthesis method using anautomatic peptide synthesizer (Model 430A, Applied Biosystems, U.S.A.)."Standard-1" was used as a program. The basic synthesis course conformedto the method described in R. B. Merrifield, Adv. Enzymol. 32, 221-296(1969). Boc-Cys(MeBzl)·pAM-p (0.5 mmole/g) was used as a resin, and thesynthesis was conducted in turn from the carboxyl terminus. Thefollowing Boc-amino acids were used: Boc-Val, Boc-Ser(Bzl),Boc-Tyr(Br-Z), Boc-Glu(OBzl), Boc-Gly, Boc-Arg(Tos), Boc-His(Tos),Boc-Lys(Cl-Z) and Boc-Ala. After the peptide was synthesized up to theamino-terminus Tyr, the peptide resin was taken out of the synthesizerand dried.

To 1 g of the peptide resin were added 1.5 ml of p-cresol and 0.5 ml of1,2-ethanedithiol, and about 8 ml of liquid hydrogen fluoride wasfurther added thereto, followed by reaction at 0° C. for 2 hours. Aftercompletion of the reaction, hydrogen fluoride was removed in adesiccator under reduced pressure. The peptide resin was washed withdiethyl ether containing 0.1% mercaptoethanol and subsequently withdiethyl ether to remove almost all included reagents. The peptide wasextracted with 10 ml of 3% acetic acid and the resin contained in theextract was removed by filtration. The filtrate was purified by gelfiltration using a Sephadex G-25 column (2.8×60 cm) (detectionwavelength: 280 nm, solvent: 3% acetic acid, flow rate: 40 ml/hour).

The fractions containing the peptide were collected and lyophilized. Theresulting powder sample was further purified by reverse-phase highperformance liquid chromatography under the following conditions:

Column: YMC Pack, A-324 ODS, 10×250 mm

Column temperature: 25° C.

Eluent A: 0.1% trifluoroacetic acid--99.9% distilled water

Eluent B: 0.1% trifluoroacetic acid--99.9% acetonitrile

Elution program: 0 minute (90% A+10% B) 30 minutes (60% A+40% B)

Flow rate: 2 ml/minute

Detection wavelength: 230 nm

Main peak fractions eluted at a retention time of 23.0 minutes underthese conditions were collected and passed through a Bio RAD AG1X8column (AcOH type, 1.8×5 cm). The washings were also collected andacetonitrile was removed by distillation, followed by lyophilization.Thus, 56 mg of a white powder was obtained. The desired peptide thusobtained exhibited a single peak at 23.0 minutes under the sameconditions as with the above high performance liquid chromatography.

The determination of free SH groups by the method of Elman G. L. Elman,Arch. Biochem. Biophys. 82, 70-77 (1959)!: 114%

Anal. for amino acids: Ser 1.65(2); Glu 2.13(2); Gly 1.00(1); Ala1.04(1); 1/2Cys 0.82(1); Val 1.03(1); Tyr 1.97(2); Lys 0.95(1); His1.72(2); Arg 1.00(1) Recovery: 74%

1/2Cys was determined by the performic acid oxidation method. The valuesin parentheses show the theoretical values.

(2) preparation of Conjugate of N-Terminal Peptide with Hemocyanin

The N-terminal peptide (5 mg) obtained in (1) and hemocyanin (10 mg)were dissolved in 4 ml of 0.2M phosphate buffer (pH 7.3), and 400 μl of2.5% glutaraldehyde cooled in ice water was added drop by drop theretowith stirring. After stirring under ice cooling for 3 hours, thedialysis against distilled water was carried out to obtain a conjugateof the N-terminal peptide with hemocyanin.

(3) Preparation of Conjugate of N-Terminal Peptide with Bovine SerumAlbumin

Bovine serum albumin (BSA) (132 mg) was dissolved in 3 ml of 0.1Mphosphate buffer (pH 7.5) (solution A). Concurrently, 11.2 mg ofN-(γ-maleimidobutyloxy)succinimide (GMBS) was dissolved in 200 μl ofdimethylformamide (solution B). Solution B was added dropwise tosolution A while stirring with a stirrer, and the mixture solution wassubjected to reaction at 30° C. for 30 minutes. Then, the reactionproduct was purified by a Sephadex G-25 column (1.5×20 cm) using 0.1Mphosphate buffer (pH 6.5)-0.1M NaCl as an eluent to obtain maleimidegroup-introduced bovine serum albumin.

The peptide (5 mg) obtained in (1) was dissolved in 0.1M phosphatebuffer-5 mM EDTA, and the maleimide group-introduced bovine serumalbumin (20 mg) was added thereto (5 ml or less in total volume),followed by reaction at 30° C. for 60 minutes. PBS was added thereto toa total volume of 12 ml to obtain a conjugate of the N-terminal peptidewith the bovine serum albumin.

(4) Preparation of Anti-Polypeptide (I) N-Terminal Peptide Antibody

The conjugate of the N-terminal peptide with hemocyanin obtained in (2)was thoroughly mixed with Freund's complete adjuvant, and the resultingmixture was subcutaneously injected into the rabbits. Thereafter, theconjugate of the N-terminal peptide with the bovine serum albuminobtained in (3) was mixed with Freund's incomplete adjuvant, and theresulting mixture was injected into the same rabbits at two weekintervals. The blood collected from the rabbits immunized by the methoddescribed above was centrifuged to obtain an anti-polypeptide (I)N-terminal peptide antibody.

EXAMPLE 11 (Preparation of Anti-C-Terminal Peptide Antibody)

(1) Synthesis of C-Terminal Peptide

Synthesis of H-Cys-Ala-Leu-Ser-Arg-Lys-Ile-Gly-Arg-OH

The C-terminal peptide was synthesized in a manner similar to that inExample 10.

Boc-Arg(Tos)·PAM-P (0.5 mmole/g) was used as a resin, and the synthesiswas conducted in turn from the carboxyl terminus. As Boc-amino acids,there were used Boc-Gly, Boc-Ile, Boc-Lys(Cl-Z), Boc-Arg(Tos),Boc-Ser(Bzl), Boc-Leu, Boc-Ala and Boc-Cys(MeBzl). The resulting peptideresin was treated with hydrogen fluoride and purified as in Example 10to obtain 200 mg of a white powder, the desired product. This peptidewas eluted at 12.6 minutes as a single sharp peak under the conditionsof high performance liquid chromatography described in Example 10.

The determination of free SH groups by the method of Elman G. L. Elman,Arch. Biochem. Biophys. 82, 70-77 (1959)!: 106%

Anal. for amino acids: Ser 0.86(1); Gly 0.96(1); Ala 1.00(1); Ile1.00(1); Leu 1.01(1); Lys 1.05(1); Arg 2.06(2) Recovery: 68%

(2) Preparation of Conjugate of C-Terminal Peptide with Hemocyanin

Using the C-terminal peptide obtained in (1), a conjugate of theC-terminal peptide with hemocyanin was obtained in a manner similar tothat in Example 10 (2).

(3) Preparation of Conjugate of C-Terminal Peptide with Bovine SerumAlbumin

Using the C-terminal peptide obtained in (1), a conjugate of theC-terminal peptide with bovine serum albumin was obtained in a mannersimilar to that in Example 10 (3).

(4) Preparation of Anti-Polypeptide (I) C-Terminal Peptide Antibody

The conjugate of the C-terminal peptide with hemocyanin obtained in (2)was thoroughly mixed with Freund's complete adjuvant, and the resultingmixture was subcutaneously injected into the rabbits. Thereafter, theconjugate of the C-terminal peptide with the bovine serum albuminobtained in (3) was mixed with Freund's incomplete adjuvant, and theresulting mixture was injected into the same rabbits at two weekintervals. The blood collected from the rabbits immunized by this methodwas centrifuged to obtain an anti-polypeptide (I) C-terminal peptideantibody.

According to the present invention, the polypeptide (I) can be easilyand accurately isolated and purified by the antibodies to thepolypeptide (I) or its partial peptides. The antibodies can assist inelucidation of the functional mechanism of the polypeptide (I), andpioneer their application for medicines. Their industrial promise istherefore high. Further, in this invention, the antibodies can beobtained by using the partial partial peptides of the polypeptide (I) asa portion of the antigen. In this case, the starting materials can beobtained by simple methods such as chemical synthesis because thepartial can be chemccally synthesized. The antibodies to the polypeptide(I) or its partial peptides can be produced more simply than the matureprotein of the polypeptide (I) whose isolation and purification arequite complicated. Hence, simple methods for detecting and assaying thepolypeptide (I) can be provided.

The following references, which are referred to for their disclosures atvarious points in this application, are incorporated herein byreference.

Japanese Patent Unexamined Publication No. 1-193654/1989 (EP ApplicationNo. 90 104 419.8 and U.S. patent application Ser. No. 07/488,696)

The Peptide, 1, Academic Press, New York, U.S.A. (1966)

Izumiya et al., Peptide Synthesis, Maruzen (1975)

Izumiya et al., Fundamentals and Experiments of Peptide Synthesis,Maruzen (1985)

Course of Biochemical Experiments 1, Chemistry of Proteins, pages 255 to332, edited by Biochemical Society of Japan, Tokyo Kagaku Dojin (1976)

Nature 256, 495 (1975)

J. Immun. Method 80, 55 (1985)

J. Am. Med. Assoc. 199, 549 (1967)

Proc. Natl. Acad. Sci. U.S.A. 80, 3513-3516 (1983)

Metabolism 8, 696 (1971)

Analytical Biochemistry 112, 195 (1981)

Nature 227, 680 (1970)

Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory(1982)

Nucl. Acid. Res. 9, 309 (1981)

Nature 303, 821 (1983)

Proceedings of National Academy of Sciences, U.S.A. 68, 2417 (1971)

Nature 302, 538 (1983)

Gene 56, 125 (1987)

Japanese Patent Unexamined Publication (Laid-open) No. 64-2572/1989corresponding to EP-251,244A

Japanese Patent Unexamined Publication (Laid-open) No.62-175182/1987corresponding to EP-225,701A

Virology 52, 456 (1973)

Science 221, 551 (1983)

Brain Research 133, 350 (1977)

Experimental Cell Research 145, 179 (1983)

Nerve Chemistry 27, 166 (1988)

Develop. Brain Res. 30, 47 (1986)

Journal of Biochemical and Biophysical Methods 10, 203-209 (1984)

Adv. Enzymol. 32, 221-296 (1969)

Arch. Biochem. Biophys. 82, 70-77 (1959)

What is claimed is:
 1. An antibody to a peptide consisting of 12 to 14successive amino acids of a sequence represented by:

    TyrAlaGluHisLysSerHisArgGlyGluTyrSerValCys,

or 8 to 9 successive amino acids of a sequence represented by:

    CysAlaLeuSerArgLysIleGlyArg.


2. An antibody obtained by using a conjugate of a peptide with a carrierprotein as an immunogen, said peptide consisting of 12 to 14 successiveamino acids of a sequence represented by:

    TyrAlaGluHisLysSerHisArgGlyGluTyrSerValCys,

or 8 to 9 successive amino acids of a sequence represented by:

    CysAlaLeuSerArgLysIleGlyArg.


3. A method for detecting a polypeptide having an amino acid sequencerepresented by formula (1):

    TyrAlaGluHisLysSerHisArgGlyGluTyrSerValCys AspSerGluSerLeuTrpValThrAspLysSerSerAlaIle AspIleArgGlyHisGlnValThrValneuGlyGluIleLys ThrGlyAsnSerProValLysGlnTyrPheTyrGluThrArg CysLysGluAlaArgProValLysAsnGlyCysArgGlyIle AspAspLysHisTrpAsnSerGlnCysLysThrSerGlnThr TyrValArgAlaLeuThrSerGluAsnAsnLysLeuValGly TrpArgTrpIleArgIleAspThrSerCysValCysAlaLeu SerArgLysIleGlyArg (1)

using an antibody to a peptide consisting of 12 to 14 successive aminoacids of a sequence represented by:

    TyrAlaGluHisLysSerHisArgGlyGluTyrSerValCys,

or 8 to 9 successive amino acids of a sequence represented by:

    CysAlaLeuSerArgLysIleGlyArg,

said method comprising: (a) incubating a sample containing thepolypeptide with the antibody under conditions that allow binding tooccur, and (b) detecting the specific binding of the antibody to thepolypeptide, said binding being indicative of the presence of thepolypeptide in said sample.
 4. A cloned hybridoma comprising a spleencell of a mammal and a lymphoid cell of the mammal, said spleen cell ofthe mammal being immunized with (i) a peptide consisting of 12 to 14successive amino acids of a sequence represented by:

    TyrAlaGluHisLysSerHisArgGlyGluTyrSerValCys,

or 8 to 9 successive amino acids of a sequence represented by:

    CysAlaLeuSerArgLysIleGlyArg,

or (ii) with a conjugate of the peptide with a carrier protein.